PROJECT SUMMARY Antigen-restricted cell:cell interactions drive adaptive immunity. While critical for understanding pathogenic mechanisms, limited techniques are available to study human immune cell interactions in tissue at sites of disease. As demonstrated in the UofC ACE Collaborative Project, the Clark Lab has developed novel technologies to identify and quantify cognate interactions in multicolor confocal images of human tissue. The current version of the Clark Lab computational approach, Cell Distance Mapping version 3 (CDM3) uses deep machine learning to identify cognate interactions between potential antigen presenting cells (APCs) and CD4+ T cells. However, limited complementary technologies are available to functionally study cell:cell interactions identified by CDM3. Historically, this issue has been skirted by examining cell subsets isolated from peripheral blood. Though some valuable insights have been generated through this approach, the subsets found in peripheral blood are not necessarily reflective of those found in inflamed tissue. To fully understand the local cell-cell interactions that contribute to pathology within tissue, it is necessary to develop novel assays that can be applied to small numbers of cells isolated from clinically obtainable tissue samples. In this grant application, we propose to use novel microfluidic techniques developed in our laboratory to address this pressing need. As demonstrated in published work and this grant application, we can isolate single cells, or cell pairs, and perform single cell functional assays for cell motility, surface expression, signaling and cytokine production. We propose to extend these technical approaches to studying MHC class-restricted B:T collaboration in cells isolated from human tissue. We hypothesize that specificities observed in the interactions between different B and T cell subsets in vivo reflect intrinsic cell differences and capacities. This hypothesis will be tested in the following Specific Aims: Aim 1: Develop methods for interrogating individual T cell-B cell interactions using microfluidics. Aim 2: Examine in situ T cell-B cell interactions in rheumatoid arthritis using microfluidics-based functional assays.